The Potential for Exosomes in the Prevention and Treatment of Preeclampsia

Abstract

Preeclampsia is a pregnancy-related complication caused by impaired remodeling of the spiral artery in association with inappropriate implantation. A maladaptive immune response in early- to mid-pregnancy is thought to allow impaired angiogenesis that leads to poor placentation. Furthermore, poor placentation can lead to maternal systemic organ damage via increased placenta-derived humoral factors including anti-angiogenic factors and proinflammatory cytokines. Exosomes are cell-derived vesicles with a diameter of 50–100 nm. They contain micro-RNAs, DNA, and proteins that can affect both local and distant tissues. Exosomes derived from syncytiotrophoblasts might promote the pathogenesis of preeclampsia, and conversely, exosomes derived from mesenchymal stem cells might ameliorate the condition. Further studies focusing on exosomes are expected to clarify the pathogenesis of preeclampsia and lead to the development of new predictive tools and treatments for preeclampsia.

Introduction

Preeclampsia (PE) is a hypertensive disorder based on placentation failure, described by a two-stage theory involving, first, the invasion of extra-villous trophoblasts (EVTs) and consequent failure of spiral artery remodeling and, second, effects of placenta-derived humoral factors on maternal distant organs (Figure 1).^1,2) It manifests as a variety of pathologies, including hepatic and renal dysfunction. In the first stage of a normal pregnancy, after implantation of the fertilized egg, EVTs proliferate and infiltrate into the decidual layer and myometrium. There they remodel the vascular smooth muscle and endothelium of spiral arteries in the myometrium to transport lots of maternal blood flow to the placenta. In PE, the infiltration of EVTs into the decidua and myometrium is inhibited in early pregnancy, resulting in remodeling failure of the spiral arteries and a reduction in uteroplacental circulation³⁾ (Figure 2). After the formation of placental villi, the surface of intervillous space is covered with syncytiotrophoblasts (STBs), and the microparticles that they secrete (STBMs) are thought to be involved in placentation.⁴⁾ In normal pregnancy, STBMs are released into maternal circulation during metabolism of STBs. It has been reported that in PE, more STBMs are released; this might stimulate a maternal inflammatory response and damage the maternal vascular endothelium, which is in direct contact with the microparticles. Extracellular vesicles (EVs) from STBs are known to impair endothelial cell function, promote endothelium-derived reactive oxygen species production, and inhibit nitric oxide bioavailability, even in normal pregnancy. Many PE-derived EVs could impair vascular endothelial function.

Figure 1.

Two-stage theory: In early normal pregnancy, immune tolerance occurs to prevent miscarriage due to the rejection of semi-allograft fetal components by maternal immune response. However, in PE, the immunotolerance is disrupted in the first stage, and EVTs, a component of the fetus, are damaged by maternal immune cells, leading to inhibition of the infiltration of EVTs into the decidua and myometrium, resulted in disturbed remodeling of spiral arteries and poor placentation. At the second stage, much anti angiogenic factors such as sFlt-1 and sEng or proinflammatory cytokines are released by damaged placenta into the maternal circulation resulted in multi organ failure through vascular endothelial dysfunction.

Figure 2.

Disturbed remodeling of spiral arteries could cause uteroplacental circulation failure in PE resulting in narrow vessels with the many perivascular smooth muscle cells. Disturbed spiral arteries cannot supply sufficient blood to the placenta by constricting the vessels. STB-exosome can damage the proper invasion of EVTs at the site of implantation. On the other hand, MSC-exosome is favorable for an appropriate placentation. EVT: extravillous trophoblast: sFlt-1: soluble fms-like tyrosine kinase-1; sEng: soluble endoglin; STB: syncytiotrophoblast; MSC: mesenchymal stem cell; eCT: endovascular cytotrophoblast; dNK: decidual natural killer cell; mφ; macrophage; Th: helper T cell. Refering to the figure of Matsubara (2021) [3]

As the placenta grows and enters the second stage of PE, placental circulatory insufficiency is accompanied by placental vascular endothelial dysfunction.⁵⁾ The number of STBMs increases further, and the encapsulated RNA, DNA, and proteins exacerbate the pathogenesis of PE. Thus, STBMs play an important role in the pathogenesis of PE. Inflammation is promoted in the placenta and systemic blood vessels due to increased placental pro-inflammatory cytokines such as tumor necrosis factor alpha (TNFα)⁶⁾ and interleukin-1β (IL-1β),⁷⁾ etc. At the same time, anti-vascular growth factors such as soluble fms-like tyrosine kinase-1 (sFlt-1) and soluble endoglin (sEng) are also increased, which inhibit angiogenesis and promote placentation and systemic organ damage due to vascular endothelial damage. In contrast, it has been recently reported that mesenchymal stem cells (MSCs) can act as immunosuppressants. One pathological condition in which they could be put to use is PE, and the favorable effects of MSC-derived exosomes on PE have attracted particular attention.

Exosomes

EVs are lipid bilayer-enclosed cargo released from cells.⁸⁾ There are three major classes of EV. Shedding microvesicles (100–1,000 nm diameter) are released from the cell membrane by budding.⁹⁾ Apoptotic bodies (50–5,000 nm diameter) are heterogeneous vesicles that are released from dying cells and cause apoptosis.^9,10) Exosomes (50–150 nm diameter) are granular materials with a surface composed of lipids and proteins derived from the releasing cell membrane, and an interior containing cell-derived materials such as microRNA (miRNA), messenger RNA (mRNA), DNA, and proteins.¹¹⁾ They begin as endosomes, when intracellular substances and cell membrane components are endocytosed, and are then released outside of the cell. Thus, each exosome reflects the characteristics of the cell it came from, and by diffusing through the bloodstream they can transmit information between distant populations of cells.

Exosomes and disease

Exosomes have been studied extensively in the context of cancer pathogenesis. Exosomes released from cancer cells create a microenvironment suitable for cancer growth (immune tolerance, increased blood flow, etc.)¹²⁾ in the area of the lesion and can act on distant organs via blood. They have also been reported to be involved in cancer cell proliferation and metastasis by suppressing immunity against cancer cells and promoting neovascularization.^13,14) Programmed death ligand-1 (PD-L1)-containing exosomes may inhibit the elimination of cancer cells by suppressing immune activity against them.¹²⁾ This suggests that exosomes secreted by cancer cells create an environment that is conducive to cancer cell growth. It is known that hypoxia stimulates glioblastoma cells to release exosomes which promote angiogenesis by upregulating protease-activated receptor 2 (PAR2) in epithelial cells.¹⁵⁾ In hypoxic bone marrow, miR-135b from multiple myeloma-derived exosomes enhances endothelial vascularization by inhibiting factor-inhibiting hypoxia-inducible factor 1 (FIH1AN) in endothelial cells.¹⁶⁾ Lung cancer-derived exosomes contain miR-23a, which increases vascular permeability by inhibiting the function of tight junction protein ZO1.¹⁷⁾ In addition, chronic myeloid leukemia cell-derived exosomes contain transforming growth factor β1 (TGF-β1), which promotes tumor growth through autocrine anti-apoptotic pathway activation.¹⁸⁾ In colon cancer, exosomes can promote tumor growth by allowing mutant KRAS to act on wild-type KRAS cells.¹⁹⁾ In addition, cancer cells and the surrounding stromal cells secrete exosomes that are involved in cancer progression; trophoblast-derived exosomes promoted invasion and metastasis of breast cancer cells.²⁰⁾ Furthermore, in breast cancer, the treatment of adipose tissue-derived MSCs with breast cancer cell-derived exosomes promotes tumor cell proliferation by converting MSCs in the tumor stroma into tumor-associated myofibroblasts.²¹⁾

Exosomes have also been shown to play a role in inflammatory disease. Gao et al. reported that mature dendritic cell (DC)-derived exosomes promote vascular endothelial inflammation and atherosclerosis by activating the NF-κB pathway via membranous TNFα.²²⁾ Adipose tissue is a major source of exosomal miRNAs in systemic circulation, and these miRNAs could regulate gene expression in distant organs and function as adipokines.²³⁾ Adipose-derived exosomes also activate inflammatory macrophages, thus increasing production of pro-inflammatory cytokines such as TNFα and interleukin-6 (IL-6) and contributing to glucose intolerance and insulin resistance.²⁴⁾ Pan et al.²⁵⁾ reported that miR-34a-deficient adipocyte-derived exosomes are largely inhibited to activate M1 macrophages and increase the production of proinflammatory cytokines,²⁶⁾ suggesting that miR-34a is an important cargo of adipocyte-derived exosomes that promotes inflammatory responses by macrophages. The extracellular release of exosomes is important for cell maintenance; when exosome secretion is inhibited, nuclear DNA accumulation in the cytoplasm promotes apoptosis via activation of ROS-dependent DNA damage response.²⁷⁾ Because blood is rich in exosomes derived from many different cell types, the analysis of exosomes in blood may enable us to diagnose and evaluate cancer and inflammatory diseases in a less invasive and more sensitive manner, and perhaps to direct treatment more precisely.

MSCs are present in almost all organs and tissues with multipotent differentiation capacity, and extracellular vesicles released from MSCs partially suppress immune activity and inflammatory responses in a paracrine manner. MSC-derived EVs are known to transmit molecules with immunomodulatory properties (e.g., proteins/peptides, mRNAs, miRNAs, and lipids) to target cells to express their functions.²⁸⁾ Zhao et al.²⁹⁾ reported that MSC-derived exosomes alleviate ischemia-reperfusion injury in the mouse myocardium via transport of miR-182, which regulates the polarization state of macrophages. MSC-derived exosomes have also been reported to inhibit inflammatory responses and promote tissue repair by suppressing pro-inflammatory macrophages.³⁰⁾ Furthermore, MSC-derived exosomes have been shown to activate autophagy and to inhibit apoptosis, necrosis, and oxidative stress in injured cells, as well as to promote cellular survival and regeneration through the delivery of mRNAs and miRNAs.³¹⁾

Maintenance of normal pregnancy and exosomes

STBs, which coat the surface of the chorionic villi, release EVs into maternal circulation through normal turnover of the intervillous space.³²⁾ STBs are the main site of EVs release during pregnancy, and among EVs, exosomes are involved in the processes of implantation, decidualization, and placentation. Placenta-derived exosomes (Pl-exosomes) are detected in maternal blood early in pregnancy and increase throughout the term of gestation.³³⁾ Normal placentation requires the remodeling of maternal spiral arteries to allow for delivery of large amounts of maternal blood to the placenta. Trophoblast-derived exosomes contribute to the remodeling of vascular endothelial cells by inducing autophagy, and endothelium-derived exosomes create a perivascular environment with Wint5a.³⁴⁾ Czernek et al.³⁵⁾ showed that cytotrophoblast-derived exosomes containing placenta-specific miRNAs such as syncytin-2 could promote regulatory T-cell (Treg) differentiation and suppress NF-κB signaling to maintain normal pregnancy, thereby reducing immunity and inflammation by inhibiting Fas ligand- and PD-L1-mediated activation of T lymphocytes and NK cells.^36,37) Tregs act as immunosuppressors. Paternal antigens in seminal plasma induce paternal antigen-specific Tregs that promote embryo implantation. Thereafter, estrogen and progesterone released during pregnancy increase Treg levels in the peripheral blood and the decidua to maintain normal pregnancy. Placental MSC (PMSC)-derived extracellular EVs are also known to increase Treg infiltration and maintain pregnancy.³⁸⁾ In contrast, reduced Treg levels can contribute to miscarriages associated with implantation failure.³⁹⁾

Helper T cells differentiate into Type 1 (Th1) or Type 2 (Th2) depending on the inflammatory needs of the local environment.⁴⁰⁾ In the peripheral blood of normal pregnant women, Th2 cells are more prevalent than Th1. It has been reported that PMSC-derived EVs stimulate CD4⁺ T cells to differentiate into Th2 cells (thereby reducing the immune response) but not Th1 cells (which would stimulate inflammation) in normal pregnancy.⁴¹⁾ This suggests that PMSC-derived EVs convert the implantation site from an inflammatory environment into an anti-inflammatory one.³⁸⁾

Another factor in pregnancy-related immunosuppression is the decidual natural killer cell (dNK).⁴²⁾ dNKs protect the implantation site from foreign pathogens such as viruses. After ovulation, increased levels of progesterone in the uterine endometrial stroma lead to a rapid increase in the number of dNK cells in the decidual layer. The number of dNK cells increase even further after implantation, but begin decreasing mid-pregnancy and become nearly undetectable by full term.⁴³⁾ dNK cells secrete CXCL8 and CXCL10 to promote migration of EVTs, and TNF and TGF-β to inhibit EVTs invasion.^44,45,46) Furthermore, NK group 2 member D (NKG2D), which is expressed on the surface of NK cells, inhibits the function of NK cells by binding to the NKG2D ligand expressed on the Pl-exosome.^47,48) In addition, the Pl-exosome contributes to maternal immune tolerance by secreting glycodelin A (GdA) by converting CD56^brightCD16⁻ NK cells into dNK-like cells in peripheral blood.⁴⁹⁾

Decidual macrophages are the major antigen-presenting cells (APCs) in the decidual layer and are involved in the local immune response.⁵⁰⁾ Pl-exosome promotes the differentiation of monocytes into decidual macrophages through the action of internalized PD-L1^51,52) and might mediate immune tolerance by reacting with HLA-G expressed on trophoblasts.⁵³⁾ Thus, trophoblast-derived exosomes might be involved in the establishment and maintenance of pregnancy by promoting immunotolerance, especially in the decidual layer.

Role of exosomes on the pathogenesis of PE

PE is a pregnancy-related hypertensive syndrome with proteinuria, liver dysfunction, and central nervous system dysfunction, and early-onset cases involve disruption of maternal immune tolerance resulted in impaired filtration of EVTs. The RNA encoded by exosomes of pregnant women with PE is markedly increased, which impairs the proliferation and invasion of EVTs.⁵⁴⁾ Therefore, Pl-exosomes may be involved in the pathogenesis of PE by disrupting immune tolerance and impairing remodeling of spiral arteries. Numbers of STB-derived EVs (including exosomes) are higher in PE than in normal maternal blood,⁵⁵⁾ and even higher in early-onset PE.⁵⁶⁾ The excessive discharge of STBMs in PE is thought to be caused by hypoxia due to placentation failure, a characteristic feature of the disease. STBMs released toward the intervillous space will flow directly into the maternal blood. The high numbers of exosomes released from STBs into maternal circulation may result in endothelial dysfunction leading to vascular constriction.^4,32) In addition, STB-derived exosomes secrete Fas-L which contributes to the pathogenesis of PE through activation of maternal lymphocytes and induction of trophoblast apoptosis. Sera from PE patients contain high levels of anti-angiogenic factors such as sFlt-1 and sEng. These factors inhibit angiogenesis, resulting in placentation failure and leading to multiple organ failure, including hypertension and proteinuria, via vascular dysfunction.⁵⁷⁾ Furthermore, these factors are highly concentrated in the exosomes of PE patients and can affect distant organs. Neprilysin (NEP), which is highly expressed in the kidney, increases blood pressure by constricting blood vessels and retaining sodium.^58,59) NEP is also widely expressed in placental villi and affects uteroplacental circulation. In PE, it is strongly expressed in STB-derived exosomes, which suggests that in addition to its role in placental dysfunction, it may also lead to hypertension and renal dysfunction via systemic circulation.⁶⁰⁾

miRNA patterns in exosomes in early pregnancy differ between PE patients and normal pregnant women, and exosome patterns of miRNA could be used for early diagnosis of PE.⁶¹⁾ miR-486-1-5p and miR-486-2-5p are highly concentrated in exosomes of PE patients,⁶²⁾ and in mouse studies it was demonstrated that PE exosomes could induce hypertension and proteinuria when injected through the tail vein.⁶³⁾ Shen et al. reported that miR-155, which is highly expressed in the plasma of PE patients, could suppress eNOS expression in vascular endothelial cells.⁶⁴⁾ PE exosomes in the serum of PE patients might induce hypertension thorough vascular restriction by reduced eNOS activity.

Maternal immune tolerance, which is necessary for the maintenance of normal pregnancy, is impaired in PE. Macrophages from PE patients disrupt implantation and placentation by inhibiting the proliferation of decidual cells through apoptosis induced by inflammatory cytokines such as TNFα and IL-1β.^65,66) Exosomes released by B cells also contain pro-inflammatory factors and are known to activate T cells⁶⁷⁾ leading to Th1-dominant in the peripheral blood of PE patients, resulted in increased inflammatory responses.⁴⁰⁾ In addition, miRNAs released from trophoblasts into the maternal circulation along with exosomes could stimulate TNF signaling in trophoblasts, thereby promoting inflammation.⁶⁸⁾ Pl-exosomes might deliver DNA, RNA, and proteins to nearby trophoblasts to create a supportive environment to maintain pregnancy, and also deliver those factors to distant organs.

Exosomes for prediction and early diagnosis of preeclampsia

Since exosomes have characteristics of many different disease-causing cells, the analysis of the contents of exosomes extracted from PE patients’ blood might aid in the early diagnosis of PE.⁶⁹⁾ Currently, PE is predicted by quantifying various proteins in the peripheral blood, but earlier and more accurate diagnosis may be possible by quantifying them in exosomes,⁷⁰⁾ where they may be more concentrated. In addition, a low level of placental protein 13 (PP-13) in placenta-derived exosomes is thought to be related to early development of the placenta as well as regulation of maternal immune responses by apoptosis of T cells and macrophages, suggesting that PP-13 in exosomes may be a useful biomarker.⁷¹⁾

Treatment and prevention of preeclampsia using exosomes

Extracellular vesicles (EVs), including exosomes, are involved in intercellular communication. They contain proteins, mRNAs, and miRNAs, and are organ-directed. These features make EVs attractive candidates for therapeutic application of PE. In addition, they offer the possibility of using a patient’s own EVs to reduce the risk of immune-related adverse events.

Trophoblast-derived exosomes are thought to act on the surrounding tissues to create a suitable environment for fetal semi-allogeneic transplantation; however, this local immune tolerance is disrupted in PE. Therefore, repairing this immune tolerance might be effective in the treatment and prevention of PE. MSC-derived exosomes contain miRNAs, mRNAs, cytokines, growth factors, and more, and can suppress pro-inflammatory cytokines that are involved in the pathogenesis of PE. They can also stimulate Treg activity, leading to the inhibition of immune and inflammatory responses through TGF-β and IL-10 production.^72,73,74) Furthermore, there is an evidence that MSC-derived exosomes can reduce oxidative stress expressed on the vascular endothelium.⁷⁵⁾ In addition, various actions of miR-133b, encapsulated in MSC-derived exosomes, support the proliferation, migration, and invasion of trophoblasts.⁷⁶⁾ This suggests that MSC-derived exosomes support EVT-induced remodeling of spiral arteries in the decidual layer and myometrium.

STB disruption leads to the release of large quantities of STB-derived exosomes, which may be involved in the pathogenesis of PE. Autophagy can protect the STBs by inhibiting apoptosis and inflammation around the chorionic villi and reducing the release of STB-derived exosomes. MSC-derived exosomes stimulate trophoblast proliferation and promote the autophagy of trophoblasts under hypoxic conditions, and might prevent PE by decreasing the number of STB-derived exosomes released through STB apoptosis.^77,78)

On the other hand, exosomes derived from immune cells contain antigens which regulate the activation of immune cells. In particular, dendritic cells (DCs) secrete a large number of exosomes. Mature DCs release exosomes that activate T cells and NK cells to stimulate immunity, whereas immature DCs release exosomes that induce Treg function and stimulate immunosuppression. The immunosuppressive effects of immature DCs make them favorable candidates for repairing the immune disturbances in PE patients. Similarly, Treg-derived exosomes can reduce immune reaction and inflammation by suppressing the induction of Th1 differentiation.

Finally, activated platelet-derived exosomes overexpress angiogenic factors such as miR-126, vascular endothelial growth factor (VEGF), basic fibroblast growth factor (bFGF), and TGF-β1, which promote the proliferation and migration of vascular endothelial cells. Angiogenesis is essential for embryogenesis and placentation. Therefore, platelet-derived exosomes may also be effective in improving the pathogenesis of PE.⁷⁹⁾

Exosomes in the blood offer promise for the diagnosis and treatment of PE.

Acknowledgements

This work was funded by a Grant-in-Aid for Scientific Research from the Ministry of Education, Culture, Sports, Science and Technology of Japan (19K09781) and a grant from the smoking research foundation (2018G024).

Conflicts of Interest

The author declares no conflict of interest.

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